Neuronal death in specific regions of the brain characterizes age-related neuronal degeneration and neurodegenerative conditions. Neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD), are neurodegenerative syndromes for which at present no cure is available. Both diseases are the most widespread neurodegenerative disorders and affect approximately 0.5% and 4-8%, respectively, of the population over the age of 50 years, forming an increasing economic burden for society.
Numerous in vitro and in vivo studies have shown a linkage between free radical production and neurodegenerative diseases and disorders, such as PD, AD and stroke as well as ALS, multiple sclerosis, Friedreich's ataxia, Hallervorden-Spatz disease, epilepsy and neurotrauma and neurodegeneration with brain iron accumulation (NBIA) disease.
Iron accumulation and oxidative stress associated therewith have been related to a number of diseases, disorders and conditions because humans have no physiologic means of eliminating excess iron.
Iron accumulation and deposition of significant amounts of iron at the neurodegenerative sites are common features in neurodegenerative diseases, and ones of the profound aspects thereof. Iron has a pivotal role in the process of neurodegeneration (AMD) (Youdim et al, 1988) as well as age related macular degeneration (Hahn P, Milam A H, Dunaief J L, 2003). Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium.
The etiology of Alzheimer's disease (AD) and the mechanism of cholinergic neuron degeneration remain elusive. In AD, iron accumulates within the microglia and within the neurons and in plaques and tangles. Current reports have provided evidence that the pathogenesis of AD is linked to the characteristic neocortical beta-amyloid deposition, which may be mediated by abnormal interaction with metals such as iron. Indeed, iron is thought to cause aggregation of not only beta-amyloid protein but also of alpha-synuclein, promoting a greater neurotoxicity. This has led to the notion that chelatable free iron may have a pivotal role in induction of the oxidative stress and inflammatory process leading to apoptosis of neurons. Iron and radical oxygen species (ROS) activate the proinflammatory transcription factor, NFκB, which is thought to be responsible for promotion of the cytotoxic proinflammatory cytokines IL-1, IL-6 and TNF-alpha, which increase in AD brains is one feature of AD pathology. This is considered logical since iron, as a transition metal, participates in Fenton chemistry with hydrogen peroxide to generate the most reactive of all radical oxygen species, reactive hydroxyl radical. This radical has been shown to be associated with protein denaturation, enzyme inactivation, and DNA damage, resulting in lipid peroxidation of cell membranes and subsequent harmful oxidative chain reactions. Such reactions cause damage to the cellular components of cells, particularly mitochondrial membranes, and therefore destroy neurons. Hydroxyl radical has been also implicated in the mechanism of action of numerous toxins and neurotoxins (6-hydroxydopamine, MPTP, kainite, streptocozin model of AD). Furthermore, such toxins mimic many of the pathologies of neurodegenerative diseases (AD, Parkinson's disease and Huntington's Chorea), one feature of which is the accumulation of iron, but not of other metals, at the site of neurodegeneration.
In Parkinson's disease, the brain defensive mechanisms against the formation of oxygen free radicals are defective. Iron concentrations are significantly elevated in Parkinsonian substantia nigra pars compacta and within the melanized dopamine neurons, wherein at the same time the activities of antioxidant enzymes at these parts of the brain are reduced. Significant accumulations of iron in white matter tracts and neurons throughout the brain, especially in the substantia nigra pars compacta, precede the onset of neurodegeneration and movement disorder symptoms.
Some of the chemical pathology of PD and AD show similarity. Apart from the involvement of increased iron, the main common features include onset of oxidative stress, loss of tissue reduced glutathione (GSH), an essential factor for removal of hydrogen peroxide, increased lipid peroxidation, the progressive nature of the disease, proliferation of reactive microglia around and on top of the dying neurons, and inflammatory processes.
Iron alone or iron decompartmentalized from its binding site by a neurotoxin, e.g. the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA), may induce oxidative stress and neurodegeneration, as evidenced in previous studies of the inventors in which intranigral administration of iron-induced “Parkinsonism” in rats and the iron chelator desferrioxamine protected the rats against 6-OHDA-induced lesions of nigrostrial dopamine neurons (Ben-Shachar et al., 1991).
The accumulation of iron at the site of neurodegeneration is one of the mysteries of neurodegenerative diseases because iron does not cross the blood-brain barrier (BBB). Where the iron comes from and why it accumulates is not known.
It has thus been suggested that treatment or retardation of the process of dopaminergic neurodegeneration in the substantia nigra may be affected by iron chelators capable of crossing the BBB. The development of iron chelators as therapeutic agents for AD and PD, as well as for treatment of age related macular degeneration has been previously suggested (Gassen and Youdim, 1999; Cuajungco et al., 2000; Sayre et al., 2000). This therapeutic approach for the treatment of PD can be applied to other metal-associated neurological disorders such as tardive dyskinesia, AD and HBIA.
Iron chelators and radical scavengers have been shown to have potent neuroprotective activity in animal models of neurodegeneration. However, the major problem with such compounds is that they do not cross the BBB. The prototype iron chelator Desferal (desferrioxamine) was first shown by M. Youdim to be a highly potent neuroprotective agent in animal models of Parkinson's disease (Ben-Schachar et al., 1991). However, Desferal does not cross the BBB and has to be injected centrally. Desferal also protects against streptozocin model of diabetes.
Stroke, the third leading cause of death in the Western world today, exceeded only by heart diseases and cancer, is characterized by a sudden appearance of neurological disorders such as paralysis of limbs, speech and memory disorders, sight and hearing defects, etc., which result from a cerebrovascular damage.
Hemorrhage and ischemia are the two major causes of stroke. The impairment of normal blood supply to the brain is associated with a rapid damage to normal cell metabolism including impaired respiration and energy metabolism lactacidosis, impaired cellular calcium homeostasis, release of excitatory neurotransmitters, elevated oxidative stress, formation of free radicals, etc. Ultimately these events lead to cerebral cell death and neurological dysfunction.
Vascular damage associated with stroke relates to elevated oxidative stress, which is caused by free radical formation and iron chelators could prevent much of the damages caused by local ischemia. Indeed, the known free radical scavengers lazaroides (21-amino steroids) have shown significant improvement of local and global ischemia damages in vivo.
Iron accumulation in aging and the resulting oxidative stress has been suggested to be a potential causal factor in aging and age-related neurodegenerative disorders (Butterfield et al., 2001). Iron chelators have thus been suggested to favor successful ageing in general, and when applied topically, successful skin ageing (Polla et al., 2003). Iron is a factor in extrinsic type of skin ageing termed photo-ageing and in non-age related skin photodamage, apparently by way of its participation in oxygen radical production. UVA radiation-induced oxidative damage to lipids and proteins in skin fibroblasts was shown to be dependent on iron and singlet oxygen. Iron chelators can thus be used in cosmetic and non-cosmetic formulations, optionally with sunscreen compositions, to provide protection against UV radiation exposure. Certain topical iron chelators were found to be photoprotective (Bisset and McBride, 1996; Kitazawa et al., 1999).
Other diseases, disorders or conditions associated with iron overload include: (i) viral infections, including HIV infection and AIDS where oxidative stress and iron have been described to be important in the activation of HIV-1. Iron chelation, in combination with antivirals, might add to improve the treatment of viral, particularly HIV disease (van Asbeck et al., 2001); (ii) protozoal, e.g. malaria, infections; (iii) yeast, e.g. Candida albicans, infections; (iv) cancer where several iron chelators have been shown to exhibit anti-tumor activity and may be used for cancer therapy either alone or in combination with other anti-cancer therapies (Buss et al., 2003); (v) iron chelators may prevent cardiotoxicity induced by anthracycline neoplastic drugs (Hershko et al., 1996); (vi) inflammatory disorders where iron and oxidative stress have been shown to be associated with inflammatory joint diseases such as rheumatoid arthritis (Andrews et al., 1987; Hewitt et al., 1989; Ostrakhovitch et al., 2001); (vii) diabetes where iron chelators have been shown to delay diabetes in diabetic model rats (Roza et al., 1994); (viii) iron chelators have been described to be potential candidates for treatment of cardiovascular diseases, e.g. to prevent the damage associated with free radical generation in reperfusion injury (Hershko, 1994; Flaherty et al., 1991); (ix) hereditary hemochromatosis and thalassemias that are currently treated with the orally-active drug deferasirox; and (x) iron chelators may be useful ex-vivo for preservation of organs intended for transplantation such as heart, lung or kidney (Hershko, 1994).
Neuronal degeneration in the CNS, particularly in the dopamine system, may be caused by an increase in oxidative stress derived from monoamine oxydase (MAO)-catalyzed oxidative deamination of dopamine and other amines. In these reactions, hydrogen peroxide is produced as a side product. In the presence of transition metal ions such as iron and copper, hydrogen peroxide is converted to hydroxyl free radical.
Brain monoamine oxidases A (MAO-A) and B (MAO-B) are two isoforms of the enzyme proven to be important drug targets for treatment of neurodegenerative and central nervous system disorders. The isoform MAO-A preferentially deaminates serotonin, melatonin, epinephrine and norepinephrine. MAO-B preferentially deaminates phenylethylamine and trace amines. Dopamine is equally deaminated by both isoforms. Monoamine oxydase inhibitors (MAOIs) act by inhibiting the activity of monoamine oxidase, thereby preventing the breakdown of monoamine neurotransmitters, and increasing their availability. Hence, selective MAO-A and MAO-B inhibitors are good candidates for use as antidepressants, for treating depression associated with various neurodegenerative diseases, for use as anti Parkinson drugs and for treatment of the causes of various neurodegenerative diseases. Several MAO inhibitors have been approved for treatment of depression and neurodegenerative diseases. For example, the MAO inhibitor rasagiline is approved for treatment of PD in several European countries and the United States. Selegiline, a selective inhibitor of MAO-B is used in the treatment of PD and has been shown to postpone the need for levodopa and spairing levodopa in early PD. However, these MAOI exhibit side effects as a result of inhibiting the catabolism of dietary amines. Sufficient intestinal MAO-A inhibition can lead to hypertensive crisis when foods containing tyramine are consumed (so-called “cheese syndrome”), or hyperserotonemia if foods containing tryptophan are consumed. The extant of side reaction varies among individuals and depends on the degree of MAO inhibition, and dosage and selectivity of the MAOIs used.
In patients with AD there is a relative shortage of the neurotransmitter acetylcholine, which is important in the ability to form new memories. Cholinesterase inhibitors (ChEIs) block the breakdown of acetylcholine, and this results in increased availability of acetylcholine in the brain and, most probably, formation of new memories. Four ChEIs have been approved by the FDA and three of them, donepezil hydrochloride, rivastigmine and galantamine are used by most physicians. There is no significant difference between these three drugs in their effectiveness in treating Alzheimer's disease, while the fourth drug, tacrine, has more undesirable side effects than the other three. Rivastigmine and galantamine are approved by the FDA only for mild to moderate Alzheimer's disease, whereas donepezil is approved for mild, moderate, and severe Alzheimer's disease. Several studied suggest that the progression of symptoms in patients treated with any one of these drugs plateau for six to twelve months, and then progression of the disease continues.
Apoptosis is considered by many experts to be an important contributor in various neurodegenerative diseases, and anti apoptotic drugs may slow down the progression of such diseases. The brains of Alzheimer's patients contain dying neurons that display some characteristic signs of apoptosis, such as DNA breaks and activation of caspases that carry out the death program. This finding leads to highly desirable new therapy strategies. N-propargylamine in known to confers its antiapoptopic activity via multiple neuroprotective pathways.
The N-methyl-D-aspartic acid (NMDA) class of glutamate receptors (NMDARs) plays a critical role in the CNS by conferring synaptic plasticity, axonal sprouting, growth, and migration. Activation of NMDARs results in the opening of an ion channel that is nonselective to cations. This allows flow of Na+ and small amounts of Ca2+ ions into the cell and K+ out of the cell. Calcium flux through NMDARs action is thought to play a critical role in synaptic plasticity, a cellular mechanism for learning and memory.
High levels of neurotransmitter glutamate and of the synthetic excitotoxin NMDA (an agonist of NMDARs) overactivate NMDARs and cause a pathological process by which nerve cells are damaged and killed. Excitotoxicity occurs by increasing Ca2+ influx into cells, resulting in activation of a number of enzymes, which damage cell structures such as components of the cytoskeleton, membrane, and DNA. Excitotoxicity may be involved in spinal cord injury, stroke, traumatic brain injury and neurodegenerative diseases of the CNS such as Multiple sclerosis, AD, Amyotrophic lateral sclerosis (ALS), PD, Alcoholism and Huntington's disease.
Adamantane and derivatives thereof are known to block the channels of NMDA receptors by acting as antagonists (see, for example, Sobolevsky and Koshelev 1998; Antonov, 1995).
Drugs with the brain as the site of action should, in general, be able to cross BBB in order to attain maximal in vivo biological activity. However, one of the main problems in the use of chelating agents as antioxidant-type drugs is their limited transport through cell membranes or other biological barriers. The efficacy of the best established iron-chelating drug, Desferal, in neurodegenerative diseases, is limited by its ineffective transport property and high cerebro- and oculotoxicity.
For this reason, 8-hydroxyquinolines and hydroxypyridinones have been proposed for iron binding as antioxidant-type drugs. 8-Hydroxyquinoline is a strong chelating agent for iron, and contains two aromatic rings, which can scavenge free radicals by themselves. In U.S. Pat. No. 6,855,711, various iron chelators have been disclosed and their action in Parkinson's disease prevention has been shown. The lead compound, 5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline, was able to cross the BBB and was shown to be active against 6-hydroxydopamine (6-OHDA) in an animal model of PD.
In PCT Publication No. WO 2004/041151 A2, various iron chelators and multifunctional compounds have been disclosed and their action in Parkinson's disease prevention has been shown. The lead compound M30 was able to cross the BBB and was shown to be active against 6-OHDA in an animal model of PD.
Dual inhibitors of AChE and MAO derived from hydroxy aminoindane and phenethylamine as potential treatment for Alzheimer's disease are disclosed in Sterling et al. 2002. Carbamate derivatives of N-propargylaminoindans and N-propargylphenethylamines were designed to combine inhibition of both AChE and MAO by virtue of their carbamoyl and propargylamine pharmacophores.
Yogev-Falach et al. (Yogev-Falach et al., 2006) disclose the bifunctional drug ladostigil (TV3326) [(N-propargyl-(3R)aminoindan-5-yl)-ethyl methyl carbamate], which combines the neuroprotective effects of the antiparkinson drug rasagiline with the ChE inhibitory activity of rivastigmine in a single molecule, as a potential treatment for AD and Lewy Body disease.
It is highly desirable to develop multifunctional neuroprotective and/or neurotherapeutic compounds as drug candidates that would posses combined functions of iron chelation, inhibition of apoptosis, inhibition of brain monoamine oxidases A and B, and/or inhibition of cholinesterase, that would also exhibit good transport properties through cell membranes, particularly, the blood brain barrier, as well as optimal oral uptake and optimal or sufficient pharmacokinetic behavior.